Several groups have been successful in the in vitro p21ras. GTP specific activation of MEK and MAPK, mimicking the biological responses observed in whole cells. Stable signaling complexes of p21ras. GTP with Raf-1 and MEK have been identified. In addition, data suggest that the signaling complexes formed between Raf-1 and MEK with p21ras. GTP are unique and independent of one another. Aim 1: Characterize the signaling complexes formed between Raf-1 and members of the MAP kinase cascade with p21ras. We will examine the requirements for Raf-1 and MEK/MEK kinase association and activation by p21ras.GTP. We will also examine the isoform specific activation of both MEK and MAPK by p21ras.GTP. Aim 2: Determination of proteins which directly interact with p21ras. (a) We will examine the ability of purified recombinant proteins, Raf-1, MEK and MEK kinase to directly associate with p21ras.GDP and GMP-PNP. (b) We will examine whether the mammalian homologues to the yeast STE5 and STE20 proteins, which are both upstream of STE11 (the yeast homolog to MEK kinase) associate with p21ras.GDP and/or GMP-PNP. (c) Identification and purification of proteins required for signaling complexes between Raf-1 and MEK/MEK kinase with p21ras. Aim 3: Examine (a) the existence and nature of p21ras dependent signaling complexes in quiescent and serum-stimulated fibroblasts and (b) the role of p21ras.GMP-PNP associated proteins in p21ras-mediated entry of quiescent cell into S phase. We will examine the presence of preformed signal complexes in cultured fibroblasts. We will also test, by microinjection, the ability of signaling proteins isolated from immobilized-p21ras.GMP-PNP to drive quiescent cells or ras-blocked serum-stimulated cells into S phase. Aim 4: Examine the potential activation of signaling proteins which interact with [21ras.GMP-PNP in a transient fashion. We have reported that the transforming p21Val12 protein appears to interact with Raf-1 more transiently than p21c-ras. We will remove the immobilized-21ras and test the activity of the Raf-1 which does not form stabile complexes with p21ras. In similar experiments, we will remove immobilized-p21ras and test the remaining protein extracts for their ability to activate recombinant MAP kinase, MEK and MEK kinase. Active samples will be separated and tested for identity with previously described MEK and MEK kinases.